Confirmation of emergence of mutations associated with atovaquone-proguanil resistance in unexposed Plasmodium falciparum isolates from Africa

Abstract

Background: In vitro and in vivo resistance of Plasmodium falciparum to atovaquone or atovaquone-proguanil hydrochloride combination has been associated to two point mutations in the parasite cytochrome b (cytb) gene (Tyr268Ser and Tyr268Asn). However, little is known about the prevalence of codon-268 mutations in natural populations of P. falciparum without previous exposure to the drug in Africa.

Methods: The prevalence of codon-268 mutations in the cytb gene of African P. falciparum isolates from Nigeria, Malawi and Senegal, where atovaquone-proguanil has not been introduced for treatment of malaria was assessed. Genotyping of the cytb gene in isolates of P. falciparum was performed by PCR-restriction fragment length polymorphism and confirmed by sequencing.

Results: 295 samples from Nigeria (111), Malawi (91) and Senegal (93) were successfully analyzed for detection of either mutant Tyr268Ser or Tyr268Asn. No case of Ser268 or Asn268 was detected in cytb gene of parasites from Malawi or Senegal. However, Asn268 was detected in five out of 111 (4.5%) unexposed P. falciparum isolates from Nigeria. In addition, one out of these five mutant Asn268 isolates showed an additional cytb mutation leading to a Pro266Thr substitution inside the ubiquinone reduction site.

Conclusion: No Tyr268Ser mutation is found in cytb of P. falciparum isolates from Nigeria, Malawi or Senegal. This study reports for the first time cytb Tyr268Asn mutation in unexposed P. falciparum isolates from Nigeria. The emergence in Africa of P. falciparum isolates with cytb Tyr268Asn mutation is a matter of serious concern. Continuous monitoring of atovaquone-proguanil resistant P. falciparum in Africa is warranted for the rational use of this new antimalarial drug, especially in non-immune travelers.

Figure 1

Detection of Asn268 mutation in cytochrome b gene in P. falciparum by Restriction digest method. 174 bp amplification products with the secondary amplification primer pair cytb2/cytb7 (lanes 3, 5, 7, 9) digested with SspI (lanes 2, 4, 6, 8) and were run on 2% NuSieve^® 3:1 agarose gel. DNA from NGATV01 containing the AAT (Asn) mutation remains uncut (lane 6), while DNA from K1 containing the TAT (Tyr) wild-type codon is digested (150 bp) by the enzyme (lane 8). DNA from patient ID011 showed a mixed infection consisting both the TAT (Tyr) wild-type digested (150 bp) and mutant undigested (174 bp) codons (lane 2). Patient ID024 had parasites harboring the mutant (Asn268) allele of cytb as their DNA remained uncut (lane 4) by the enzyme. Lanes 1 and 10 represent the low molecular weight DNA ladder (New England Biolabs, Beverly, MA) used as a marker for the electrophoresis

Figure 2

Multiple sequences alignment of cytochrome b gene (residues 137 to 279) of some Nigerian isolates of P. falciparum. Highlighted are residues 268 with amino acid changes from the wild-type tyrosine (Y) to the mutant asparagine (N) allele associated previously with atovaquone resistance in Plasmodium falciparum. In addition residue 266 in patient ID (Cytb012) where Pro (P) is changed to Thr (T) is also highlighted. Sequences of the atovaquone resistant (NGTV01) and sensitive (K1 and 3D7) control strains are also present.